Polynucleotides encoding human estrone sulfatases

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

The present invention is in the field of enzyme proteins that are related to the sulfatase enzyme subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is therefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the sulfatase subfamily.

Sulfatases

The novel human protein, and encoding gene, provided by the present invention is related to the sulfatase family of enzymes, including estrone sulfatases. Specifically, the novel human protein of the present invention is a novel alternative splice form of a gene provided in Genbank gi5689491 and published PCT patent application WO200055629 (see the BLAST and Genewise alignments of the sequences of the present invention and the sequences of gi5689491 and WO200055629 provided in FIG. 2). The evidence supporting alternative splicing includes a different polyA signal used to create the protein of the present invention compared with the art-known protein; the stop codon at cDNA positions 1223-1225 and polyA signal at cDNA positions 1750-1756 are present in the genomic sequence; and the last exon of the cDNA of the present invention crosses the splicing site of the corresponding exon 5 of the art-known protein (these differences are illustrated in FIG. 2 in the BLAST and Genewise alignments of the sequences of the present invention and the sequences of gi5689491 and WO200055629).

Novel human sulfatases, such as the protein provided by present invention, are particularly useful as targets for treating cancer, particularly breast cancer. Sulfatases are important for generating estrone and 5-androstenediol from sulfated precursors. As stated by Purohit et al., (Mol Cell Endocrinol 2001 Jan 22;171(1-2):129-135), “The development of inhibitors to block the formation of estrone and 5-androstenediol from sulfated precursors is an important new strategy for the treatment of breast cancer”. Thus, sulfatase inhibitors are useful for treating cancer and, consequently, novel sulfatase proteins are valuable as novel targets for the development of anti-cancer therapeutic agents. Purohit et al. found that non-steroidal and steroidal sulfamates, particularly a tricyclic coumarin sulfamate (“667 COUMATE”) and 2-methoxyestrone-3-O-sulfamate (2-MeOEMATE), inhibited estrone sulfatase activity and “offer considerable potential for development for cancer therapy”. The importance of sulfatases relating in breast cancer is further described in published PCT patent application WO200055629, “Novel Methods of Diagnosing and Treating Breast Cancer, Compositions, and Methods of Screening for Breast Cancer Modulators”.

Enzyme proteins, particularly members of the sulfatase enzyme subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of enzyme proteins. The present invention advances the state of the art by providing previously unidentified human enzyme proteins, and the polynucleotides encoding them, that have homology to members of the sulfatase enzyme subfamily. These novel compositions are useful in the diagnosis, prevention and treatment of biological processes associated with human diseases.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human enzyme peptides and proteins that are related to the sulfatase enzyme subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the enzyme protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain.

FIG. 2 provides the predicted amino acid sequence of the enzyme of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the enzyme protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a enzyme protein or part of a enzyme protein and are related to the sulfatase enzyme subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human enzyme peptides and proteins that are related to the sulfatase enzyme subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these enzyme peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the enzyme of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known enzyme proteins of the sulfatase enzyme subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known sulfatase family or subfamily of enzyme proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the enzyme family of proteins and are related to the sulfatase enzyme subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the enzyme peptides of the present invention, enzyme peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the enzyme peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated enzyme peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. For example, a nucleic acid molecule encoding the enzyme peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the enzyme peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The enzyme peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide. “Operatively linked” indicates that the enzyme peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the enzyme peptide.

In some uses, the fusion protein does not affect the activity of the enzyme peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant enzyme peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the enzyme peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the enzyme peptides of the present invention as well as being encoded by the same genetic locus as the enzyme peptide provided herein. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a enzyme peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by the same genetic locus as the enzyme peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

Paralogs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the enzyme peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the enzyme peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a enzyme peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant enzyme peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as enzyme activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the enzyme peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a enzyme peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the enzyme peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the enzyme peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in enzyme peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Accordingly, the enzyme peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific, assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a enzyme-effector protein interaction or enzyme-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, enzymes isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of enzyme proteins, particularly members of the sulfatase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to enzymes that are related to members of the sulfatase subfamily. Such assays involve any of the known enzyme functions or activities or properties useful for diagnosis and treatment of enzyme-related conditions that are specific for the subfamily of enzymes that the one of the present invention belongs to, particularly in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the enzyme, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the enzyme protein.

The polypeptides can be used to identify compounds that modulate enzyme activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the enzyme. Both the enzymes of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the enzyme. These compounds can be further screened against a functional enzyme to determine the effect of the compound on the enzyme activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the enzyme to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the enzyme protein and a molecule that normally interacts with the enzyme protein, e.g. a substrate or a component of the signal pathway that the enzyme protein normally interacts (for example, another enzyme). Such assays typically include the steps of combining the enzyme protein with a candidate compound under conditions that allow the enzyme protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the enzyme protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant enzymes or appropriate fragments containing mutations that affect enzyme function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention farther includes other end point assays to identify compounds that modulate (stimulate or inhibit) enzyme activity. The assays typically involve an assay of events in the signal transduction pathway that indicate enzyme activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the enzyme protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the enzyme can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the enzyme can be assayed. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Binding and/or activating compounds can also be screened by using chimeric enzyme proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native enzyme. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the enzyme is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the enzyme (e.g. binding partners and/or ligands). Thus, a compound is exposed to a enzyme polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble enzyme polypeptide is also added to the mixture. If the test compound interacts with the soluble enzyme polypeptide, it decreases the amount of complex formed or activity from the enzyme target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the enzyme. Thus, the soluble polypeptide that competes with the target enzyme region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the enzyme protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of enzyme-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a enzyme-binding protein and a candidate compound are incubated in the enzyme protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the enzyme protein target molecule, or which are reactive with enzyme protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the enzymes of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of enzyme protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the enzyme pathway, by treating cells or tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. These methods of treatment include the steps of administering a modulator of enzyme activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the enzyme proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the enzyme and are involved in enzyme activity. Such enzyme-binding proteins are also likely to be involved in the propagation of signals by the enzyme proteins or enzyme targets as, for example, downstream elements of a enzyme-mediated signaling pathway. Alternatively, such enzyme-binding proteins are likely to be enzyme inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a enzyme protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a enzyme-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the enzyme protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a enzyme-modulating agent, an antisense enzyme nucleic acid molecule, a enzyme-specific antibody, or a enzyme-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The enzyme proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. The method involves contacting a biological sample with a compound capable of interacting with the enzyme protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered enzyme activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254 -266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the enzyme protein in which one or more of the enzyme functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and enzyme activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. Accordingly, methods for treatment include the use of the enzyme protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the enzyme proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or enzyme/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the enzyme peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a enzyme peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the enzyme peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the enzyme peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the enzyme proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in enzyme protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a enzyme protein, such as by measuring a level of a enzyme-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a enzyme gene has been mutated. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate enzyme nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the enzyme gene, particularly biological and pathological processes that are mediated by the enzyme in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain. The method typically includes assaying the ability of the compound to modulate the expression of the enzyme nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired enzyme nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the enzyme nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for enzyme nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the enzyme protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of enzyme gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of enzyme mRNA in the presence of the candidate compound is compared to the level of expression of enzyme mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate enzyme nucleic acid expression in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for enzyme nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the enzyme nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in pancreas adenocarcinoma, breast, colon, and brain.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the enzyme gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in enzyme nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in enzyme genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the enzyme gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the enzyme gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a enzyme protein.

Individuals carrying mutations in the enzyme gene can be detected at the nucleic acid level by a variety of techniques. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a enzyme gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant enzyme gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the enzyme gene in an individual in order to select an appropriate compound or dosage regimen for treatment.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control enzyme gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of enzyme protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into enzyme protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of enzyme nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired enzyme nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the enzyme protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in enzyme gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired enzyme protein to treat the individual.

The invention also encompasses kits for detecting the presence of a enzyme nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in pancreas adenocarcinoma, breast, and colon, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting enzyme nucleic acid in a biological sample; means for determining the amount of enzyme nucleic acid in the sample; and means for comparing the amount of enzyme nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect enzyme protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the enzyme proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the enzyme gene of the present invention.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaninated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified enzyme gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fuasion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroenzyme. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include YepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kuran et al., Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as enzymes, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with enzymes, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a enzyme protein or peptide that can be further purified to produce desired amounts of enzyme protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the enzyme protein or enzyme protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native enzyme protein is useful for assaying compounds that stimulate or inhibit enzyme protein function.

Host cells are also useful for identifying enzyme protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant enzyme protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native enzyme protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a enzyme protein and identifying and evaluating modulators of enzyme protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the enzyme protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the enzyme protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, enzyme protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo enzyme protein function, including substrate interaction, the effect of specific mutant enzyme proteins on enzyme protein function and substrate interaction, and the effect of chimeric enzyme proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more enzyme protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

6 1 1799 DNA Human 1 tatttcattt tagtctcacc gtctccgttt ttctctgact gcccagaact ccagaaatca 60 ggagacggag acattttgtc agttttgcaa cattggacca aatacaatga agtattcttg 120 ctgtgctctg gttttggctg tcctgggcac agaattgctg ggaagcctct gttcgactgt 180 cagatccccg aggttcagag gacggataca gcaggaacga aaaaacatcc gacccaacat 240 tattcttgtg cttaccgatg atcaagatgt ggagctgggg tccctgcaag tcatgaacaa 300 aacgagaaag attatggaac atgggggggc caccttcatc aatgcctttg tgactacacc 360 catgtgctgc ccgtcacggt cctccatgct caccgggaag tatgtgcaca atcacaatgt 420 ctacaccaac aacgagaact gctcttcccc ctcgtggcag gccatgcatg agcctcggac 480 ttttgctgta tatcttaaca acactggcta cagaacagcc ttttttggaa aatacctcaa 540 tgaatataat ggcagctaca tcccccctgg gtggcgagaa tggcttggat taatcaagaa 600 ttctcgcttc tataattaca ctgtttgtcg caatggcatc aaagaaaagc atggatttga 660 ttatgcaaag gactacttca cagacttaat cactaacgag agcattaatt acttcaaaat 720 gtctaagaga atgtatcccc ataggcccgt tatgatggtg atcagccacg ctgcgcccca 780 cggccccgag gactcagccc cacagttttc taaactgtac cccaatgctt cccaacacat 840 aactcctagt tataactatg caccaaatat ggataaacac tggattatgc agtacacagg 900 accaatgctg cccatccaca tggaatttac aaacattcta cagcgcaaaa ggctccagac 960 tttgatgtca gtggatgatt ctgtggagag gctgtataac atgctcgtgg agacggggga 1020 gctggagaat acttacatca tttacaccgc cgaccatggt taccatattg ggcagtttgg 1080 actggtcaag gggaaatcca tgccatatga ctttgatatt cgtgtgcctt tttttattcg 1140 tggtccaagt gtagaaccag gatcaatgta cgtatttctc tgtttgcaac attcaactgt 1200 cgtacctcaa gtgtgtctaa gataattcaa ttaccagtct cagtatctgg tttcctttca 1260 tccaaaacaa aaaaggatgt gtgtaggctg gttaatttcg aagatgaaaa ccttttcctc 1320 cctgccacat cttaaattag ctcaagtata ctacttaaag agaaaggaaa aataagtgta 1380 tcaatgacta attctctcaa attgactgga atctatgtct ttttggtctg tgtgcacaga 1440 caggatgtga tcttctggga tatcaccctt ctttgaatca gagatacgct gtcatttaaa 1500 aaaaaaacct gacaccatcc ttttagtgtt taacttttaa aaattattcc gaaagaaatg 1560 tttttaaaag ataaattttg aaaagctggc ttttctttta aaggaaaaag agctaaagga 1620 ctaggctgct atttctgtca ctgtaggcag gtcactgctt ctctttgcat ctctattttc 1680 ccatcatgaa atggccttgc ctattttccc atcataaaat ggccttgtca atcatctcag 1740 gatgttttga ataaaatggg attgcatcca tgaaagaaaa aaaaaaaaaa aaaaaaaaa 1799 2 372 PRT Human 2 Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu Gly Thr Glu 1 5 10 15 Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25 30 Arg Ile Gln Gln Glu Arg Lys Asn Ile Arg Pro Asn Ile Ile Leu Val 35 40 45 Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser Leu Gln Val Met Asn 50 55 60 Lys Thr Arg Lys Ile Met Glu His Gly Gly Ala Thr Phe Ile Asn Ala 65 70 75 80 Phe Val Thr Thr Pro Met Cys Cys Pro Ser Arg Ser Ser Met Leu Thr 85 90 95 Gly Lys Tyr Val His Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys 100 105 110 Ser Ser Pro Ser Trp Gln Ala Met His Glu Pro Arg Thr Phe Ala Val 115 120 125 Tyr Leu Asn Asn Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140 Asn Glu Tyr Asn Gly Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu 145 150 155 160 Gly Leu Ile Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170 175 Gly Ile Lys Glu Lys His Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180 185 190 Asp Leu Ile Thr Asn Glu Ser Ile Asn Tyr Phe Lys Met Ser Lys Arg 195 200 205 Met Tyr Pro His Arg Pro Val Met Met Val Ile Ser His Ala Ala Pro 210 215 220 His Gly Pro Glu Asp Ser Ala Pro Gln Phe Ser Lys Leu Tyr Pro Asn 225 230 235 240 Ala Ser Gln His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Met Asp 245 250 255 Lys His Trp Ile Met Gln Tyr Thr Gly Pro Met Leu Pro Ile His Met 260 265 270 Glu Phe Thr Asn Ile Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser 275 280 285 Val Asp Asp Ser Val Glu Arg Leu Tyr Asn Met Leu Val Glu Thr Gly 290 295 300 Glu Leu Glu Asn Thr Tyr Ile Ile Tyr Thr Ala Asp His Gly Tyr His 305 310 315 320 Ile Gly Gln Phe Gly Leu Val Lys Gly Lys Ser Met Pro Tyr Asp Phe 325 330 335 Asp Ile Arg Val Pro Phe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly 340 345 350 Ser Met Tyr Val Phe Leu Cys Leu Gln His Ser Thr Val Val Pro Gln 355 360 365 Val Cys Leu Arg 370 3 42571 DNA Human 3 gtatcaggtt tctcacgatt taaaacaaat gcacagaaac caaacagtca gtgcagaata 60 attgcaggct ttcagtgttc agcatgtaca gcaatcactg tggaatcacc ctgcgttatt 120 aagaagaaag caccaaatct tacattagtg acttctacag ggctgcgtta tcaattggag 180 ctgtcttgtt tgttgcagat aatgtagtca ggactgcctg gctgcagaca ctagagtttt 240 gtttaaaaac cgatttcttc ttgtctcttt ctctctcttg cagtataatt acaggctgca 300 gagtgaaaag cattagaact gtttacaaaa cagctcataa agtttaaaat aatggggata 360 cgtgtgtgtg tttgtgtaaa acaaaataat gtgtatggta ggggtaaaca atatccagtc 420 tttcttcttt cactaccccc tgtcaccttc cagaattaag ggcatgaagt tgagagatgg 480 agccctttcc tcctgctatg cgatgcttac acttaattag ttatgcctac ttatccaatg 540 ccagtttatt gttgcagatc aaaatacaga ttctcaggtg tatggggact gagtggctga 600 tgaaacagac tgctatctaa ttaattttag ggcagcctaa attcccataa agatgttccc 660 tcatgacata tgagaggaag attttatttt tttaatgagc cctttgctat ctttccaaga 720 gaaaagcttt cagcaggtta gtgttccaaa gtgagagggg catttttcca accctttcaa 780 aagcctcctt ctgtgcagct ttgcaaagat tttgcagctc gcccttctgg attttattta 840 tttatttttt aatgcggaag ggtagccgct gactccagcc tcggggccaa tcaatcattt 900 tgctttgcag gtttaagatc tgtgacaaag cgaaacccct gtgctatctg tgccttacca 960 gtctcaccaa caataagcct ggtgactgac aatcgagagg gggctctgtc cacgtaggtg 1020 ccggcacaag ctgaggacat gagtgggaca gaggaaccag ccttgcacgg aggaagcacc 1080 ttttccttct ggtgattgat tgatggggga cagtgaggag gttttcagag actggaaaaa 1140 attgtcccag tcacttacta tgaagtcttt gtcagcagaa aagactctcc ggggtagaga 1200 atgatataat gcagatgaca aatgacaggt gtgtgtttcg ttctgcttgc taggtactca 1260 gtatcacacg caggtgagtc agcgctcccc aacatgcccc ttgcgccatc tgctccccac 1320 atgcaaacac tcgttcccaa cgctctgtgg tttccctggc actgctggct cttcctaatc 1380 gatcgtcagc tctgttgggg atgtgtaaag tactgtcaga gtgtgagcag ggtgatacct 1440 taccaccctt ttatggagct gattatgaaa tgaagatagc atttgaatca tttgttagca 1500 gttctgaaag ttgtttcctt ctgttcctcc cttttggagc acagaagaaa aaatatatgt 1560 aatatataca catataatat gctgttgcaa gagactactt cagatcgaaa atctgttttt 1620 aaaatcattg actgatattt cctttgtatt tttttctccc ccttccagga ccctatctgc 1680 agatgttctg aatacctctg agaatagaga ttgattattc aaccaggata cctaattcaa 1740 ggtattagct ctcgtcagaa agcttttaca tttgagctct gtgttggaaa ttctattttg 1800 gcaatgaatt gaaataggaa aagttggaat gagaataaag gacaaaagtg aatttgcaaa 1860 ataatcaagt gcttaaaaaa ctacccagca cttgtgaggg tttgctattt ctgactcatg 1920 tgcaaccctg tctctgccag cttatgtgcc aatactgact tatttgtagc cctttctctg 1980 caactgtgct tggagtttgg atttcatttt agtctcaccg tctccgtttt tctctgactg 2040 cccagaactc cagaaatcag gagacggaga cattttgtca gttttgcaac attggaccaa 2100 atacaatgaa gtattcttgc tgtgctctgg ttttggctgt cctgggcaca gaattgctgg 2160 gaagcctctg ttcgactgtc agatccccga ggttcagagg acggatacag caggaacgaa 2220 aaaacatccg acccaacatt attcttgtgc ttaccgatga tcaagatgtg gagctgggtg 2280 agacactgga ctcttcactt gttagtctct tttgttcaga tgatttctcg agtctcagga 2340 ttatcaggag acattctgag gctttgcact taattattgc acattaacca acaccctagt 2400 ttacgcaatg aacttgtatt gaccataagg catttggttt gtgtttcagc attacttttc 2460 tgatgttatg cttttgaaat ggtcggggaa ggggcctggg ggagtaggac aatggagaaa 2520 gagggtcagc actgaagact gtagaaggaa aggattgaaa gccctcagtt aagacattgt 2580 aaaaatattt gggcaaagtt gtttcaaaga gtatgaggat gtgactgtaa ttttatgcaa 2640 tggatatgaa tatagactga tactaaagga actttcagtg gttattagta ttagagtgga 2700 ttacttattc acagtttgtt atagtaattg ttaggtaatt caaagttgca gtgttctata 2760 tgtcttttgg tagagaatcc acttactact accttagata tgatgctttt ttatttagct 2820 tgcctaggct aagcgtagag cacccagaaa gcctgccaaa atctagtgat tctaacttac 2880 cttctatatc acctgactgg gtttcttacc ttctcaccgt cttcaatggc ccagccctac 2940 agtcttgttc ataagccaag ggccaattct tctagtccac ctagtgcaag gcagatagaa 3000 agcttgcccc tagaagttgt cactaccact cctcatttct tttcctgaac ccaaattcct 3060 tgctctcagg catcacccag ctgtgcttag ccatcacatt caacctgact ggtagttgaa 3120 tcttctagca gagcatgctg ggcttcttta ccgagctcct gaggctcagg ttcttgagga 3180 taaaactctt cacgctggca cttggtctcc atggaagggg actttgcttt cccacttgaa 3240 accagacggt gagatcccag taaagttaat tccttgggtt cagctggaag caaatgcgct 3300 aaaaagccag cagatgtcat tattgctgac gttggtttga ggagtcaacc caactttttt 3360 ttttttttaa caagggtatt gattttcagg cgacaggcca aaatgaaagg tgtcacacat 3420 acatgagtgt gtatttagca catatgatgt tagtatgtat gtaagtggtg gtttaaatgt 3480 tttcattcac ttacagagca agtaatttta gcttttttag agccttgtgg gtccatttca 3540 agttagttta gtgcctaatg tgttaatagc acagtctctg catgaggatt gcaatgttaa 3600 acatatcctt gccctctgct tgacctcaca cctgaactca ccttccttaa tatctcaccc 3660 atccatcgct tttgctacag ctgagatctc tggatcctct catctttccc agtttttccc 3720 tcaccggttt gtcacctggc ctgcctgcct cctctagtct tggcctctct tgcccaccct 3780 tcactcaatt gccagagtta tctttcaagt atctctctga tcagatcact tttctgctta 3840 agtcccttct gtggtttccc tttgccatga gattatgccc ttctcctctg tgggcaaatg 3900 agttctgagc cttgcgaccc ctgcctgtcc ccaaggctgt ttccctttcc tccactttca 3960 ctctgtgctt cataaacaca attgccatct ccccaaaggt gtctggtggc ttcacccccc 4020 tcgcctctat ccatgccttt gcacatctca tcccctctgc cttttcctct ttccccacct 4080 ggagaaaccc tacctgttct tcataaccca gttcatgtca tgcgcttggt gttcccaaaa 4140 caaaaccacc ctcccctcca gcagcattga tggaactttc ctttgcaccc cgagaacacg 4200 actccagcat ggtgctcatt ccaccatagc cttccatgcc tgtcccttcc acttgaccaa 4260 gatcaaccag agagcaaggg tgtgttttat gttgcactcc tcatgcactc ctagtgcctg 4320 ggaatatagt gggcactaaa ctaacttgaa atggacccat aaggctctaa acaagctaaa 4380 attcccagaa ataaatataa gtattcatca tcatctacct cttcaatata cagcactgtc 4440 tttaaaatat attaagaaat tgctcataac tttctttttt aacagaattc aaattttcag 4500 ccttgtccta tagttcattt attcatccag taaatatttt ctgagcagtt accaagtatt 4560 gccttctgtt ggaggcatgt gactatcatg gtgaagggtt atagaccagg ctcagccaac 4620 ccagatattt cctacatatt tgcctttaac tccttccttg attttctagc cagataacat 4680 gctaagaact agctcattgg ctgggtgtgg tcattctcgc ctataatccc agcacttcgg 4740 gaggctgagg caggcggatc acctgaggtc aggagttcaa gaccagcctg gccaacatag 4800 tgaaaccccg tctgtactaa cagtacaaaa attagccagg catggtggca ggcacctgta 4860 atcccagcta ctctggaggc tgaggcagga gaattgcttg acccgtgagg cagaggttgc 4920 agtgagccga gatcacacca ttgcactcca gcctgggcta cagagcgaga ctccatttca 4980 aaacaaaaaa agaactagct tgtttaactc tcacagtaac tctaggaagt agttactatt 5040 ctctctttaa tttaggtatg agaaaaatga ggctcagaga agtcatgtag cttgtggtga 5100 gcaagtaaat tgtgaaaccc agacctgtat tagatctgag tctccacagc tacgtattta 5160 accactgtcc cttccattaa tcagccatct acaaataatt attaagcacc tcctaaaaac 5220 aatcggcaaa ttcataatat tttaggttct gggagagagg gaacagaagc aaaagatagg 5280 ccactggcaa aaacaaaatg actaggaacc acagtgaact gtgggttgga ggtccagaaa 5340 agacctcctt tcttggcaga aaagtcatgc tctcggtagt ggttcccgct tgatggagac 5400 ttttcctcat ttttctgtaa tgtgctcata atccttctag aacattattg ctctagattt 5460 tgggtgttgg ttgttttgtt tttgtttttt taccatctta accattttta agtgtacaat 5520 tcagtactgt taagtacatt tacattggtg tgtaacccat ctccagaact ctttccatct 5580 tacaaaagta aaaccctata cccattaaac agcaactccc gtttctctct ccctcaatcc 5640 tggcaactat cattctactt tctgtctctg tggatttgac tactctaggt atcttatatg 5700 ggtgaaatca tacagcattt atctttttat gactggctta ttttacttac cctcatgtcc 5760 tcaaggctca tccatgttgt aacatgtgtc agagtgtcct tcctttttaa tgctgaataa 5820 cttttcattg catatatgta ccacattttt ttaacccatt catccactgt tggaaatttg 5880 agttgcttcc tccttttccc tattgtggat aatgctgctg tgaacatggg tgtacaaaga 5940 gctcttcgag accttgcttt tagttatttt ggagctatac ccagaagtgg aattgctgga 6000 tcatatgata ctcttatttt tattattttg aggaaccacc ttactgttct ccatagcaac 6060 tcaccttgtt acgttccctc caactgagca caagtgtcat aatttttcca catccatgcc 6120 cacacttgtt attttctggt tttgttttta atagtagcca tcctaatggg catgaagtgt 6180 tatctcattg tcactttgat atggttaggt tttgttttgt tttttactcc agtgcaattt 6240 tctttgccaa gtgctttaat aaatcttcac ctaatgcaaa cctttttgca cctgtaatgc 6300 tgtaggcaat tttggctact gtgcctccaa aaagaaagta acagacttag taaagactta 6360 gaagaacaat tatgaaaatt ctaatagaat acttttctca ctgggaaagc ataaaacaat 6420 cctatctgtt tatctagaaa gatgaaggct gagaagggct ataatcaaag tttataaaat 6480 cgagccaggg aggaaagtac tagttcatta tgagcaggag cccttccctt cactttcagg 6540 acaaataaac tattactctg taaagggagt aagacacata aggaattcat taataaaagt 6600 actagaacct tgcatatgga taacacttta tcttttccca aagaacattt tgttcacatt 6660 attgtgaatg aatattgcca tctcacatga gttgggtggg taggacaaag atcatcatcc 6720 caggaggaaa cagcctgtgg aaagatgaaa tggcttaacc atggtcccct tacttctgaa 6780 tttgcttttc ccacaactta agaagttgtt cacgttgtgc ctatacccgt aagtacttca 6840 gagttttagg ctagaataat gggtggaaga tttacaattt tttaagctaa gtaaacacgc 6900 aaggaagtga tcaacataaa gttcaggata atggctacca ctggggaaaa agtgatgacc 6960 gagagcatca gtacattcac attctatttc aaaactgaaa aaatatatat ctgaaagaat 7020 ataatcatgt taagttttgt taaagctagg agatgcgtta tttattctgt tacaccctac 7080 acttgaaata ttttataaat cagaaggaag aaaataaaaa atgtttgagg gactcctaac 7140 tttctgagcc tagcatcaaa gaaattgggc tctaaatctt tcccaaatgg cctttggtgc 7200 tagtctgaga aacaaaatac tgagcagaat agaccattga tctgactcaa catggcattt 7260 cttacattcc taggaaattg acctgacagg aacatctcag taacattgac ccgatgtttc 7320 tcttagatca tatgtatagg gaagctatga gtacatggag atattttgtt tctttaaaac 7380 aaaagccaga acacaagata ttcagaccag agctagccca cagatgtatg ttgtttgatg 7440 agcatacttt tcagaattaa gccaatattt taaaatcata ttttaaaata tatataacat 7500 ataaaaaaat cagattctga cgtttcttga aaattcaaaa aatgaaggct tgtattccca 7560 tgaagcagtc atcacttgga tgtttgaagg ggagctgcag ctgctctcgg acactgggga 7620 tcccccacta ctgtgttgtc cctcaataac ttatgccaag gtgcaggtgc catttatcat 7680 tgaccatact gctgtttttc ttatagtcag gaaaagctct ataaaccatg tctctatcaa 7740 aggtaaaaca acaaaaagac acaaaaaccc tctgactttt cttatacctg gctgccttca 7800 cttgcctacc ttatctatgt agccctgtag gcatgttaac ttccattcct gtaagtagct 7860 tttaatggcc ttgtgctctt ttggaggggc caggaggaaa tgatgagtct tagtgatctg 7920 gaaacattcc agactaattt attatcttct cttttttccc ttcttctccc aactaccttt 7980 tccccctttg gttttaggta aaaaacatct tttttttttt cttgccaatt ccatagtttg 8040 ggtagaaaaa aaataccatt taattttgtt attttttata tgcttatgct tttagaatta 8100 gaatattcaa tagatcatac aggcatttta ataaaggaaa tgtcactgtt tcatgtttca 8160 tactttacta tgaaaataac tgttggcatg tgaaaatggc agggaagtac ctaatacaaa 8220 cgacttaaat atttaaagca ttagccttac ttcatcataa aagaatgaca tcgacaaaca 8280 cccctccaca atcagtaaga gctgggtttg cttgttgttg tcaatcttca aaccatgaat 8340 gctctaatcc acatggaagt atctctctag gaagccaggt gtatatgaat cccacatgtc 8400 tgggatttcc ataagtgata atgacaatat ttactgttga atttctggtt ctgcgagttt 8460 ttctggacat gacaaacagg tttgatgaaa tttttaatgt ttagtaatac aaaatactgt 8520 atactcaaat gctaagatat atgtatcggg aaaggtaaag ctttgttttg aagtttaaac 8580 atgtcaatga agtaattcag gatctccgtg tgattttagc aaagtcatcc aaattagggg 8640 gacattttct ccgatccttt ttcatacaaa tgatttttgt atgaaatcat tctgtatgaa 8700 atgattttgg gaatcaagta gttggagtat ataaagtgat ttaaattcct cactatggtt 8760 ctgtctagcc acaacctatc acatactcca tccaaagcac tattcctgga gctttgattt 8820 ttaactgtgc caaaattgcc aacatggtgg atcttgatgg attcctgaag ggtggcactt 8880 ctcccactcc cagccaccgc tgacctcttc agcatcctgg tccacagccg atggctgtca 8940 cagcatccgc ctgcaggagc aatggaagat gttctcagtc ccagcctgga actggggcac 9000 tgggacagtg tgtcttggca gtcctgtggc ctggaccttc cctctcatca agccagccca 9060 gtcagcagtg catgcacacc agctacacag acataatcac ggttgcttca gagcatttgt 9120 gtctggctcc cacttcatgg gacaacttaa aataacaatg aagttgggct ttttgcttag 9180 ggtgaggagt cagtgtgtgt agaaaaggga agaattgata tttgtatttg aaaaccaata 9240 tcaacatttt aagtcttaca gcatagaaaa ggtagttgac ctctgttctc tcctccagct 9300 tttggacttc agatgtagat tacagagaag aagatggaag aggcaggaga aaatttaatg 9360 tgatggggtt gaggtgagca catagggcaa ccacagccac ctcggcacac tccttacagg 9420 aagtacttga tggcatcctg ccgtttgctc agcaacaacc ttgtaaacta gtcggagtgg 9480 ggagcgctat tagaagctcc attttacaga tgagaaacac aaaccaaaaa aggataaatg 9540 acttgctcag gctaagctgt ggcaaaatcc aaatgttttc ccacatgttc aagtctaggg 9600 ctctttccct tctcatcttt atcatcctca cccctgcctt gtgaatctga ttatgagtgt 9660 tcagctttac aattcatatc attcctatga aataattgtt gtaataactg agaatcagca 9720 ccgtttgcat ttcaggcacc caaggcctga aacctacaga agtgaagagc ttcagcttaa 9780 aacaaaactg agcccttcca cttatcggac agacattgtc ctgtccactg ctctcaacag 9840 ctgtgaataa ataactatga gttttattac agaatcattt ttagtaaggt ttgccaataa 9900 aaaaatcaat ggcacaatca cagtgggaat atatacaggg taaggcagtg ttattgactg 9960 acattaatca ttgcacagcc attaaaaggt gctcgatgct ttccttactg gtacacacta 10020 acacagtgga gtgaagagat tcagattcct gcacaccgtc atctgtctct acccagagaa 10080 tccatagaga tcagaggcaa caattaacgc caccatggca gtcagcagct aaagacagac 10140 agatccacag cccactcatg cctcatctgt gcctgagcca ctgagaccat ttcagccttg 10200 aggggagggt aggagacagg tgttaatgtc agtcagtatc tcctctaaga ggcgagaaga 10260 atcaaaagat gagagtgcag aaagagagac ctatcgcgac tgcaagcaga cgtcaattct 10320 gtttctcatc ttgaaaatgt caatgtcaca gccagcattc agcctagata gagctcaaga 10380 gcatggtctc tgggaagact gcctgcattt acttctgggt tctgctactg agggtctgtg 10440 tggccctagg cacagcatga tttagccttc tgcacctcca tttcattatc tgtgatatag 10500 gaataatagc tgctagcacc caccacaaag gattattgtc tggatcaaat taattgctca 10560 tgtgaagcac ttagaagaga acctggctga gagtaagtag aaaatcgatg tttctttttc 10620 ctgtctacaa tataccttat agtcttggca gtgatctaag ccacctagat cactcaaaat 10680 taggcaccca ctacagtctc ttccagctct aggcttccct gattctgtgt ccaacctaga 10740 atactttttc aggtgagatt cactgagagg cagcttcggc ttcacccagg tttcattaga 10800 aacacaaatt ctcaggcccc atcctggact tactaagcca gtatcttggg gtgggtacag 10860 gattctaggg cttattatgc ccttcaggtg attttttaaa gttggacaag cattgtctca 10920 gaatctcttt actgagaacc ttcaagccag attcccactt tttgttttgt ttttatgaga 10980 cagagtctcg ctttgtcccc caggctgaag tgcagtggcg cgatcttggc tcactgcaac 11040 ctctgcctcc tgggttcaag caattctcct gcctcagcct cccaagtagc tgggtctaca 11100 ggcgcatgcc atcatgctgg ctaagttttg tattttcagt agggacaggg ttttaccatg 11160 tcggccagac tggtcttgaa ttccccgcct caggtgattt gcccaccttg gcctcccaaa 11220 gtgccgggat tacaggcatg agccactgag cccggcccga gattcgcatt ttcaaggacc 11280 ccagtggtgg agctgtattc ttaacagtgc ctatggggat ccactgttgt cttggttcct 11340 tttgacgtgt ggactggtgg gagccagggc atctggaggg attgcttggg agccctacac 11400 aatcagctca gttagttaag aaaaggatga tactgagaat gagaggagaa tcctagctgg 11460 ggcacttcca gcagtgatga attgggataa gttttagccc ctgtttttat attattttga 11520 ctttaaatca gatttctacc tcggtcccat atttgtagtc taaatgagtc tgtaaactag 11580 agattacagt gctgcgattc agaaccttgg atgatggcct cagctgcatt tttcttttat 11640 ttgaattctg ctaaggctca taaaggaaac tgggagcttg ttctttgaaa tagactactg 11700 gacactgaaa agccatcaac agggtttgct gttttcccca aaatcaagat actcagtaac 11760 taactactgc agagatctaa agaggagcaa tgagacatgg tagaaatctt cagtgtcacc 11820 tttcaccctc tttccacttc tacatctccg ttcatccact tatctccagg atattccccc 11880 tgtgtgatca accaacacaa atataaaatg caacatttct aaaatggggt tttaaaatct 11940 cccataaggc atattccttt ttagttactt cttcaccatc agtgaaacga ttggtttcca 12000 gttttcttct cacggtgaaa acttcaaggt gttagccaac tcccagcctt ctcatgcatc 12060 accacatcac ttgggtctgc ttcttagggt cgctctgtgt cagaggcata gtcgtccgag 12120 ttcttctttc ctcaggggta gacggcttca gcgacctcat ctgcaccctt cgtggaccag 12180 cagcagtgtg ccctcctcaa tccacacacc accattgcaa ctgcaacgcc tctcgagggg 12240 attgcaatcc actgcagcag caacacctta caaagacttt tttcttaaca tgccgttcgt 12300 gtctttgaca cagaacataa ctctaccttt gtttttttta ttctatcaca tccagtatct 12360 ctgcccagcc ttcaagctat ttgctaaatc tgctttccac atttcatgaa gagaattctc 12420 catatttctc ttcatgccag gaaatcaatg tgttgttctc atcactctac ttgtttcttc 12480 ccacctccag agcatcacac atgtcactcg ctgcaatccc ccaatgatat acttcaccca 12540 tccgacattt acttcctcct ttttttccat ctgtcctcat ctctcatttc agaactctta 12600 ttaagccatc aatccaagtt ccctctattt ttatatgccc tgctactgct gtattcacat 12660 tgttacgtgt gcattgcttt gcaaatatcc ccaagtgatt tttctatgta ggttctcctc 12720 tagagaattt attactgaag accagaggca gaaaatttcc catttctttt gtttatgcta 12780 cacttgggag atcagcccgg aatgctgagg ttgatagaat gttttgcatg aagtaggcac 12840 tcaagacatg ttattcattg actacaaaag gttgcttgat gccctgaaga caaccagaag 12900 agaaagagca gttaggattg ctcctagccg aggcatattt tagggagctc taatgagacc 12960 caattagaaa ccctcctcct gttctcatat gcgtcatgtg gaacggtaaa agctagtata 13020 tgaagacacg gggctaactg tggctttttg gactgttgca tgcttctctt ctgccctgaa 13080 tgcttgcctg gtttgcatta cgatttcctt ccttgctgga aaacatagac tttcttcctt 13140 tctttggaag aagtgggacc tgttggaaat ctgaggctcc aagattcaaa tgcaatgggc 13200 ttggctgtag tgctctgtag tacacttctt gacaagtcct gaaaagacta ttgaattctg 13260 gcctgtgctt ttctgcagtg tttggatgtg tttcagctgc agtgtttggc tggacagagt 13320 aatcgaaata gttttttttt ttttttcctt cctcagcaat cttacgttac cagttgatcc 13380 ttaaagttaa aatggaatat tttacaaccc tgcaaatact ttcgagtgca atcgaattat 13440 agctctttca caagcaaaca gcctatcttt aaaaaattgt gcataaatag gtcaaaatat 13500 aaattgatgt tgttatccta atagaaaaac tggcaaacat ttggtgagct tgctagagga 13560 taccaacttg cattgaagat cttttttaat tattattaaa caaacacagg cattttgatg 13620 ggacttaata tggaagaaat ttttttctct tttctttcat acgattaaaa tgctatagta 13680 gtaatctaca ctagtaatct agtagcaatg tcactagtag attgcatttg tgctgggctg 13740 ggggttttga tgctctgtgt atcaggctat tgttctctgg taatcttcaa agggctgctg 13800 gcctttgaat ctgctccata tctaaaattc tagctttaat tatcagatta gcctgcattt 13860 ttttttcttc gtgattacag ggaggagaag tgtatgttta ttattctgca aaatacttga 13920 gatggtagga tctcaaatgc ttaattttgt tgtagaaatg agagctgatc aaattataga 13980 ctcctttaag ctaaggaaag gaaatgaata agtcagggaa acagaggcac tgaggggcac 14040 aatcgaaata ggcattcatg tgctgcactt tgctaaacaa tgctggcact gtgcctttca 14100 gggtccctgc aagtcatgaa caaaacgaga aagattatgg aacatggggg ggccaccttc 14160 atcaatgcct ttgtgactac acccatgtgc tgcccgtcac ggtcctccat gctcaccggg 14220 aagtatgtgc acaatcacaa tgtctacacc aacaacgaga actgctcttc cccctcgtgg 14280 caggccatgc atgagcctcg gacttttgct gtatatctta acaacactgg ctacagaaca 14340 ggtaagggat gacgtttcta gcccatgaac gtcttgtaat atgtcttaga ctcaggaaga 14400 agtgtcatgt aatgaaatgc atgaagttcc aacaaatacc taaaaaagga tcaagtgttt 14460 ttaaactgct tgacatctac cccagggtct gggacacagt aaatgatcaa aattatttat 14520 tatttattaa ttaatgaaaa actgatgggt aaatcaatca tgtgtacctt gttgattcat 14580 gtatttgttc attcataatt aataggattt ggaaagtttc tttagcttgt ggttgttttt 14640 aggtctcaaa tgttcacttc taccttccag gaaaggaagt catcctaatt gctacacctc 14700 ctattattgt ttaacctaca cagggcaaga agaggttttt gattaattgg ttttttgaaa 14760 attggggact tctttcaaga ggggtagtga acatcatgcc agtctttctg agaaaaaagc 14820 aaggttcctt ctggtaatat tagccccaag gccctatcct cccagcatgt agatgatgtc 14880 cttgggtttt ttgtagcatt tcttcataaa agggcacaac gttgttcgta gaagggctac 14940 agtgcagaca tgggttgttg cttgttttta tttttccaga atcacctagg cttattcata 15000 agaatcaaat atgttatgat taaagcttgt tcaaatgact ttcaggccta caatttctct 15060 attttgaaga tttaaaagta aaggagttca gataaatatt ctcctgcatc tcttttaaaa 15120 gtgaaattaa tctttcccac tggactcagg aaatatatgg ccatttgctt ctttgcagag 15180 cgccctacgg gcacttaata cttgttattt atggatgaaa atattgattg tgcatatgat 15240 agcactgtca ctcgcagaca gctcaaagtc tcgactcgaa gcagctcctc ccgtgcatct 15300 caagggtgtt ttcttatgcg taggaaagaa actaggttac gtagaggtta cagagagtgc 15360 cttatctgag tgtgttttct cacattagtg atttaaattt atagcacctt tcatttgagg 15420 actctaaagc aatttgcaaa tccaccaaac atccctgtag agttcataga tggcaaatgt 15480 acttggctct gttttataac tgaggtgggg atttcaaata ttgcgtgacc agctcagaaa 15540 cagaatcaga ggcggttccc taataaccgg ttcctgattt taaccatcaa attgtgtttt 15600 gcttctggga agtgttgtgt gaaatcagtg cctttcacct cccaggttcc ctagtagcaa 15660 acatggaact ggaacgattg ggactctcac atatccagag tgaaatggat gacatttgtc 15720 ataagaaata gagattgagg gtgagacagg gccctgccag gacttagagc agtcaactag 15780 ggtttgctaa tttgtttcag ggctaaatta ggaagggcaa gaaagaaggg aactccattt 15840 tgccattatc tcacctaatt cctagggcaa tcttgaaaga aaggaatttc ccatgttact 15900 aatgaaaaaa ccgagactca gaaaggctaa ttgtctatgg tcatgagcta gtcggtgatg 15960 gaatgaaccc agcacatctg actccaaagt ctttcctctg ttcattgtct tatttattta 16020 tgtaatgatt ctcttctgcc tcattccaaa acgccttcca gagatacaga ctttagaagc 16080 tacagaccca tgaggaccct ggcaaacaga aacaaagagt aggactagct aaagtcaagg 16140 caaagattgc tgtacaaact gcttgctgtg gagttgtagg gaaaggtgga atatttagaa 16200 ctaagcatcc agatgttaag agcaaaaatg gaaacaccat ccattgtaaa gttcacccag 16260 tcaccgggat aaaaacaatt cagttgcctc taagcatgtt tcctggtact tgcttcggtt 16320 atactgtgct atatccacct taagagggat tgagagagtt taactaaaac tcaaagatga 16380 tttagttcca tttccttatt tttagaggag caaatttaca cccacaagaa taaagtgact 16440 gaagtgacag atctgactgt tttttattta tttgtttatt tattttattt tattattatt 16500 atactttaag ttttagggta catgtgcaca atgtgcaggt tagttacata tgtatacatg 16560 tgccgtgctg gtgtgctgca cccattaact cgtcatttag cataaggtat atctcctaat 16620 gctatccctc ccccctcccc ccaccccaca actgactgac gttccaatgg gaagagccgg 16680 caccgggacc ttagtcttca gggctctctc cagtgtgctg atcaaacctt aagaatcggg 16740 tctgggatga aactgtgtac acaccggaag gcttccctgt tacctcaatg gactgtcact 16800 tttttgtgca gcccgagaag tttgtttcag tcgatgtctt ctccagggag agtagatttg 16860 ctcctggaaa ctaaatctgt ttctaaacct tttccccagg gtttagacac ttaggtaata 16920 atacaattta tagtgagtca acctccaaaa taaaaggaat tctttaaggg aaataaagac 16980 ttttccaaaa acttagttaa cagtaggaaa aggggctggg tgcggtggct cacgcctgta 17040 atcccagcac tttaggagga caaggctggc ggatcacgag gtcaggagat cgagaccatc 17100 ctggctatca cggtgaaact gtctctacta aaaatagaaa aaattagcca ggcgtggtgg 17160 cggacgcctg tagtcccagc tacttgggag gctgaggcag gagaatggcg tgtacctggg 17220 aggcggagct tccagtgagc caagatcgtg ccactgcact ccagtctggg agacaaagcg 17280 agactctgtc tcaaaaaaaa aaaaaaaaaa aaggaaagaa aaaccagtag gaaaaggata 17340 atatgggctc aggaaaacaa atctagattt gcctattagt aaatcagaaa aaataaaaat 17400 tggggccggg tgcagtggct catacctgta atcccagcac tttgggaggc tgagatgggc 17460 gtatcacctg aggtcaggag ttcgagcctg gccaaaatag caaaacccca tctctactga 17520 aaatacagaa attagccagg tgtgttggtg catgcttgta atcccagcta ctcaggaggc 17580 tgaggcagga gaatcacttg aaccaaggag gcagaggttg cagtgagcca agatcgcacc 17640 attgcactcc agcctgggca acaaagcaag actctgtcac acacacacac acacacaaaa 17700 aaaaaaaaaa atgggaagac tgaaatggca gtgatagaga tctctataat ttaatttatc 17760 cttgatggga aaacattaca gtatctcaga caattaggaa gtaaaaatgt cttcatgaca 17820 atagttattg atccaaccat ggacaacaac ccatttgaaa atacacaaat aagtacacac 17880 aagtggcagg aatcttagaa aattaagaga attggtaggt agatattaaa ccaatgtaat 17940 tatgagacac taaactagta gggagaaagt caatgtgtaa agattctatt gctttcctaa 18000 taatgaattt attatttgga acatttagct acaaacctaa gaaaaataat gacggaaact 18060 gaaatatgta ctatacatac ataaatatcc atctagagag taagttttat gtagaataac 18120 tcagagaaaa caaattacta aagggcttat tttgggaggc gtccagaatc catggattta 18180 aatgagttga attaatccaa tattatattt accattgtat gacatgagtt ttctctttca 18240 gaactgaggc atatttttgg actggctata gcctaaattt ttctaagttt actcatggaa 18300 aatatgagtc tatgagagct tgaatgttca agaaggggaa aatgcagtaa catgtcgact 18360 gcacttcata ttctgacaag tgaaatagga atgagattgg attatatatc ataaaaatga 18420 ttatcctgcc tgaaatcagc tccaaaaaaa ttaacttaaa tttattatga ggatatcaac 18480 attaatataa aacctggctt aggttttaag gtaaaaataa ttatccatgg ttgagtgata 18540 gcattaagta tttaacaaca acaaaaaaaa ctgaagcaag attgaaaaca tcgtggaatc 18600 acattctacc ttgatttgtt ttgaggactt ggactggcca gttttccaat ctgtatgatg 18660 cagagattgg cctagaatga ttcctaagtt ttctttcttg tccaaagtta cagtgtgtta 18720 taactttatt atactcatgg taattataat attgattcat ttaacaattg tggtatttga 18780 attataatcc taacacatgg tacataaaat cacatatgag ttaaatctta atcacatgaa 18840 ttctacctca catcactgag gtagaaggga caatatttaa tagacagctg caataaatat 18900 ttttgtgaag aattcttttg tcataaatca aaatcataga ccttagttct gaaaaagaaa 18960 aattatatca aataatggta aatacaatat tggatcaaaa atttttttgt ctttttacgt 19020 tacaaaattt atgttactta tagcaagtat atttattctc taatatgaga ttttttaaat 19080 gtagagttca cttaaagtaa gacaaactaa tattcttaat tttattatga tgtagattct 19140 tgatacatgc ataaacatga gaaatgtcat acatttatta aaccacagtg tgcttggaac 19200 acactagaca ttgggatgaa gacatttaaa ggaagattcc tgtcttcact agacttacaa 19260 tctagttgag gaaaccagac tgctgtatac aaactgacat taatcataat tttaacttgg 19320 ttcaaaatta tttatattta tagttactag cgtgaaatcc atcaccctaa gtctatcaat 19380 tacgtggatt aaaatctcaa tatatctttt gatacattaa ataagatttg acttttctgc 19440 ggcatcagat ctttgggtta gtcactattg ctggctttaa aagaaattcc ttggcttcag 19500 gtagttcctg gaaatttttc taagcattat ggaacaggtt gtcctagaca gaagtagcat 19560 ggcctgaagc caacaataat tacaatcagg tcttctgatc tttctccctg ccccccaacc 19620 cccaccacct tcttaaacag ctgtgaaggg aagtgcttaa tggtatccaa aacaaagagg 19680 atgggtaaat ggcacattag tgatgtattc agatagtagg agttgaattg aattgccaat 19740 gccgaaggat agaaaaatat tgaactatac gtaacctaca tgtagacata atggcagtaa 19800 gggcaagaaa gctaaattca ccttaggaag ggaaaaagag atttaataca tctggaggaa 19860 aataattaga aggccagata atcaattgca gagcgccgcc aggaaacatc gtgttgaaag 19920 aggccggggt gattacaaac gagtctcaat gtcatgaggc aacaaaaagg ccagagcaac 19980 tggaggccaa cagtgctgca ccctgacacc caaggccccc atcagccttg gaatgagtgt 20040 gatgggtgag cgcacatctg gaatactgag tacatttctt acgctccgtt aacacagaga 20100 caccaaaaac ctggagggag ttctgaagca aataacaagg actattaaaa gacttgaagg 20160 aatagtttat aagggaagga ttaagtcaaa ggggaatgcc atggcaatgg gtagaaaaca 20220 gtacaaaata tcctacaaag taaaacaatg aggaaagggc aggaatgact tggggagagg 20280 aaacaaaaaa accccaacaa tgaagtaact taaaagtgca gaaaaaaaaa ttaaaactaa 20340 ttaagcagaa aaatgtaagc caaatggagg agtttgttgc cacaaaataa gtagtagtgg 20400 ggaagaaaat atgtaacctc cgaagagata ttttcaagtg cacaagtgca gaactctagt 20460 gcgagatttc cttacactgc aggatggaaa atcatttaca aaagacaggg ccaaaagaat 20520 actgctaatg gtgatgctaa taacaatatt agttgtagga gcacttaaca agccgttgtt 20580 ttgtgccagg cactgttttc agcgctttac atatgtgttg atgcatttaa tcctcaaaac 20640 aatcctgcca ccattattat tatcaccata gtggctttgc agaaggggag ttgggggagg 20700 gagaagtgaa gtaacttgca tgtagatgga taccctagca agtagcagag ccagaatttg 20760 aacccaagca ggctggctct agggtttata ttctcaatca ctatgctttt tgccttcttg 20820 gaaaaaaaaa aaaaaaagga aagaaaagtg ggataaaccc gtagggatga ggaggaggcc 20880 aaggaaagca cggggcttga ggctgttaag tgcaagcttt ttggaaacaa tcgcttttga 20940 acgttagtgg ggtgtggcct tggtctgctg cctgtggctc caagtcatac tgcattttgt 21000 tggaaaagga aaatcatctt gtggttctat gtgaaaaggt cagttcgtct ctaagacagg 21060 aattcctcat taaaagaatt ccaactacac gtagtcagca cagaaggaaa tcctgagtca 21120 cctgatgtga gaccctttga cactttgccc tacactgatc aacgtgctca gtgcccctgg 21180 cagaatgctt aagcagcggg cacttggctg actgtagacc taattggttc actcattcac 21240 agagccaaca aataaacatt cattcaacaa acaaacattg ccatgtttct cagactggag 21300 tctagattct tttaaaaata atataataag aaataacaat tttagaaact ctaaagctct 21360 attctatgaa aatgttttga aggccaaatc agctttaaaa aatatgatga tttgattggg 21420 cgcagcgcct catgcctgta atcccagcac tttgggaggc ctaggcagat ggatcgatca 21480 cctgaggtca ggagttcgag accaacctga ccaatatggt gaaaccccat ctctatttaa 21540 aaaaaaaaaa aaaaattagc caggtgtggt ggcgtgtgcc tgtagtccca gctacttggg 21600 agtctgagac aggagaatcg cttgaaccca agaggcagag gttgcagtga gccgagatca 21660 caccactgta ctctagcctg ggcaacaggg taagactcca tctcaaaaaa ataaaaaaga 21720 taattcaagc aaaatcacaa aatttttaaa gtctagacct cgtaaagtcc ccagaataca 21780 ttggattcat gaacccaaat tcaagaaaca aaaaggatgg agccctgaac tgtgtgcaag 21840 gatggagagt gcctcagaga taaggcagag gcaatgtttg ccctcaaaaa gcttacagtc 21900 tagcaggtgt tcagcttcta tatgaacatg actataccac aatggagaaa gggaagatga 21960 cattacaacc acaaagacag tgttgtggaa ttaagacaga gactgtgagt gaaatggcat 22020 ctgctctggc cttgatatat aaagaggcaa ataaagagaa ttgcacaagc aaaaatagag 22080 aggtgggaac cagagagcaa atagaggaaa cattagctgg agagagggac gattaacaga 22140 gaaataggag atggggttgg aaagggaagg attttgtcca aactcaaagt aggcctctga 22200 gggcaagcta gcagagtaca cttgattctg caggcaatga gggtaatctg agattgcgag 22260 aagagggtga agtaaccaga gcaggtcctt gggaagatta accagtggca atcgaagtgg 22320 aaagaccctc catcctggct gggaaagtca gtgagaccat gagcatctgt gaggggtggc 22380 aagttaccag ggatggcagg agggatttga gcactatttc caaggcagac ctgataggag 22440 gtggcaactg cccagcaagg ggagcgcagg agcaggcgaa agcagagggg gctctggagg 22500 gccaagcatg gttcatggag ggtgattatg ccattcagag gaatggagta aggctgaaag 22560 ggaaaactgg taattccatt ttaggcaatg gcattaggaa gcaagtaaaa cattcagatg 22620 gaggaattct acaggtacag gtgctccttg acttacgatg gggtgacatc ccaataaacc 22680 catcataagt tgaaaataag gtaagtcaaa aatgcattta atatactgaa cctaccgaac 22740 accagagctt agcttagccc agtctaactt aaatgtgctc agaacactta tattagccta 22800 cagttgggca aaatcatcga acacaaagcc aataggcttg taataaagta ctgaataact 22860 cataggattg attgagtact gcactgaatg cataccaatt ttacaccact gtaaatgtga 22920 aaatcttcaa ttgaaccatc gtaggtcagg gaccatctgt agtcgtatat caaagataaa 22980 agcaagctaa atacctatcc catagagaca tcaaaattat gtacatcatt aagtttaaaa 23040 ttcagaatgt gtgttttaag accaatgtca ataaagtgct gcaattctag aattcgtttc 23100 tattatccca agccagtctt ccaggaacta cttttttacc atggatataa gcgagggcac 23160 ctataaaatc tgtttaatga agccaggcat tggctttgac atggaaggcg tctggcaaca 23220 gctttataac atcagaaaaa ctaaaacttg cctacatatg tatatgcatg cataggggtg 23280 tgtgtgtgtg tgtgtacaca cacatatata catacacgca cacatgctta tacctataca 23340 gacatatata aaataaagtt ttctctagcc ctttctactt gaagggaaag ctatgtgtgt 23400 ggctggagtg actaaacatt taggtttacc cagaatcatg cttgtttata cctgcttttc 23460 tgtaattaca gcaattacaa ataacatcct cttgctctct caaaagtatt ccagtatatt 23520 tatgactacc attctgctag gttgtaatgt ctttttcact tcaagaatga acccatattg 23580 ttcctggaat cccagcttct tctttgcttc ccgtacccct ctcctgtcat catcttttgc 23640 agaagaccaa atttctagtc accctctcag agagaccgag tcagccctgt ggcacagtgg 23700 tctttcttgg aagtgacatg ccaaagttat aaatgtgaag gccttccagt ggctttttta 23760 gtgaactgtg gtgtctttgt gacacataca cttctactat actataattg tatgaaaatt 23820 agtaatctat gtagtaactc tatgttgaca gaatttttat tatcgataat agatgtatac 23880 attcataaaa tacacataac ataaacaccc attacatact atacatgtga tataaaccct 23940 gaccatatcc cataaaaatg gagtttacca tggttccctg gtttggaaaa tttgtactct 24000 ctggatatgt aaaaacgaaa ataagctttt caatagtgtt tttataattc acaattctca 24060 aatagtaagt tagaaaactt atcacaaaga ctgaactttc agttctccaa cacctgcccg 24120 gtggttgcat tccaaatctc acgctacttc tctgattgtt ccatcaactt aacaaaagag 24180 catagcctga ttttactcca gtaggaccat aagaaatgaa tgcacccaga gtgctgtgat 24240 cattatgatg gtttcattga gctgtaatcc atgtacttgg atactacttc tatttatttt 24300 ttaaaaatgt gtttgtgtca ctttgccaaa ggattggagt attacactaa tgtcattttg 24360 gcattcacta ttacctaggg caacttttgt tttaccgtct ctttttcaag tcataatttt 24420 atacttatcc atttatttat gattaatcat tttacgtgaa aaaaataatt cttttttccc 24480 actgcagcct tttttggaaa atacctcaat gaatataatg gcagctacat cccccctggg 24540 tggcgagaat ggcttggatt aatcaagaat tctcgcttct ataattacac tgtttgtcgc 24600 aatggcatca aagaaaagca tggatttgat tatgcaaagg taattttcag gcacttttac 24660 actgcatcaa tttactttgt gcataatggg gaaaagccat tttcagtgag ttaaactatc 24720 cacaagattg gctttctatg ttctcacaat gttagcatga gaaatgttaa ggtaatttta 24780 aactctaggc aaggaaaaga ctctcaagga acgctgcctt tgtgtagtga tttccctcat 24840 taggatgaaa ggcaatcagg ctttgatgaa agtatcatca agaaaatcag aattctctgc 24900 tctcttatga taatttttgt cctcccagtt cccccggacc caaccaagga cttgtccaca 24960 taatcaaatg ttcatcttgt actgttttac ttttcactgg gacaaaagta tattttgtct 25020 gtggcttcag atttaggcac aagcataaga gcaaataaat atgataatta aagtttgaaa 25080 aaccacattc cttgctttta ctcctgtctg accaagctta gtatacgtga caaggacacc 25140 ttccctatca cggcaagcat ccacaaaagt ctctaatgct atcaattcta ggattttcaa 25200 atcagttcag agaaactgaa atcaacatgt cccatagttc tttgaccagt gggttctagt 25260 tttgacttaa aaattcacaa agattttgtg atagctgact taagtttaaa tttttttcaa 25320 atcataagaa tgaaggggaa aatatcttcg aatttagcat gcttatttgc caaaatatcc 25380 ccttcccttc cagccatacc catctcttct tcatttatct agaagaagcc gagaatctgc 25440 tctatctagc aacctctccc aacaggctag atcacttggt agaaatcgga aggagagaac 25500 ttgatttaat gttggcatat tgctgtcttt atgcttggcc tgatttgagc acaagggact 25560 tgatgggaga taagattaag tccagctcct ttataccctt cagaaaacaa tgaatgcaaa 25620 tgaattcata aacattgcta ataggcttcc aaactcatga aagttaaaag ttagcagaga 25680 ccttggaggc aaatttgagc aatgtcctca tttgcaaaga tgagaaaaaa gcattctaga 25740 gtggctcaac cactcatcct aggtcatatc cccagctgtg gatacaatca ttgcaagcaa 25800 atggtgcaga ccacgtgcac taattgtcac tgctctcctt tgctgtctgt agggatgctt 25860 tttcatgctc cttgttcaag ttattaacct ttctttccct gctgtcctaa agagagcaaa 25920 gtaatcaaga ttctctccaa atactaaatc agcgtaactt gttcattatc acagctgatt 25980 aagtgtcaaa gacaactgtg tctgaaaaga atatatatct ttttttagtg aggaaaagaa 26040 tgaaacagac actcccttgg aagaggaagg ggatagcctg tagacttgcc ctaacaatga 26100 catgcggcac acaccatccc tctgatactg cttttgcagc tgttctggtc cttaaatcca 26160 caacatgtga ttagccatgc ctggaagcct tcaacatttg caaatattgc ctaaacactt 26220 tctgaataaa gtttatactg gagctccaag ccaatgacac acacttaaaa gaagcaggtg 26280 gtttaagttt tcatcttttc tttccttttc attccatttc ctccctccct cttacagacc 26340 tgcatcagcc ccccctgact gtgggttaag tcattttatt agcaagtcag gctctaatcc 26400 cagcagctgt attgctttag ttgtgcaatt aacacagtat aatctgcagg aaatcaactg 26460 ctccctattc aagtgtttca agtaaattaa ctgatcaaat gttgcagctt ttccctgtgc 26520 tcctggattt tggccatggc tttgattact gattattgta attcccacag gtggattttt 26580 cgtttgaaga aaatatcttt tcttgtgttt atgtattcat gggcgtgtgt gtgtgagcgt 26640 gtgtgtatgt atgtgtgtgt gttctgcaac tgtaaatttg aagtgggcgt gggtgtttcc 26700 tgcccttaaa gtaattaaat tttttgccaa ggaattacat caatgaaacc tgagactgaa 26760 atatgtatcc ggtgtttcat gtgttctagt acttttatcg ccagattaat cattatcttg 26820 ggcaaacacg acttgacttt ttttttcccc attgctaagt tgtgtattac ttaaaatcca 26880 tttttcgtat gttaccaagc tagcaaccct agaaaacaac tggcagctga ttttctctat 26940 tatcgaaaat gttcggctgc cttgggaggt gcagccttcc ttcctgctgt agaccttgcc 27000 acttcgtgca gtgaattgct tctgaggaaa gcagttattc aaatgcgatc tgatgaatgt 27060 caccttttgt aatttttgtt ttgtgtcaaa tgtatgtttc aggactactt cacagactta 27120 atcactaacg agagcattaa ttacttcaaa atgtctaaga gaatgtatcc ccataggccc 27180 gttatgatgg tgatcagcca cgctgcgccc cacggccccg aggactcagc cccacagttt 27240 tctaaactgt accccaatgc ttcccaacac atgtaagtaa caaactcaac tctgcgacct 27300 gccgaacatg cctttccctt ttctcctcat cccactcctc tcctttaccc cgtttccttc 27360 caccctgcgt atccacaagg ctttcttcat gaaaggataa cttaagagca gaccacggaa 27420 caggcagagc cgctgagcct gaaagaaagc gccttatctg ggtggtttga ggaggaatca 27480 aatttccagc atttacaagt agctaaatag aaaggaagag atgcacatag agtgaatggg 27540 ggcaagtttt acaagagttt cctttcgttg tcttaaataa tattcgtgtg tctgatctaa 27600 taatgatgat gatcaaatag tatgcttttc atagctgcac agtggggacc tctggtctgg 27660 ttatagaaac atggatttat tttccaggcg aataccgtag cagctttgct gcagacgtgc 27720 aattagaatt cctgcagaag gcagcttgag tggcttgccc aagagggctt ctcaggtcac 27780 agctttaaaa taacctgatt tttttttttt taaagaggca ggagtcttgg agatgggggg 27840 tgggaaggca caagggagag ggctgatggc gtggagggat gagacagaac aaagagctgt 27900 cgtgtgccca caattctcac cagccaaagg tggaaaaatc tagatgcttt ggcagcaaag 27960 aacatgattt tgttgttcac tcagttgaca ccatttcttc ctaagctttg ccatcaatat 28020 ccagtcttcc acacagagca gtggagttgg ctctgtgtct gctgaaagcc tgaccattag 28080 ggagacaggg aacagaaaat tggtatctgt ttcctatatt gtgaaacctc caaaattggt 28140 tcttaatcta tttgtactta aatatcatct cttttcatcc acactggtta ttagccaaga 28200 ttccaggcag aaagaacctt acgaaaatag gtaagtaact atgcaggctc tctagttgcc 28260 ggtcactata catccctaga gaagttttta taaaatgttc tctttttttt gagacagagt 28320 cttgctctgt aacccaggct ggagtgcagt ggtgcaatct tggctcactg caacctccgc 28380 ctcctgggtt caaacaattc tcccacctca ggcttctgag gagctgggac tacaggcaca 28440 cgccaccaca cctggctaat tttttgtatt tttagtagag acgcagttcc accatgttgg 28500 tcaggctggt ctcaaactcc cctgacctca agtgatccac ccacctcggc ctcccaaagt 28560 gctgggatta caggcatgag ccaccgcacc cagccttata aaatattttt atttgtacct 28620 taatgtaact gattgactta tgactcctgg tcagtggtac acagatcatc tctatgatat 28680 catgtgactt agaccagaaa gaaggaggcc agagctgact caggacaaga actaacaata 28740 tgaagccagg gtgggttacc tactgagcat gcccaggaac tcagaggatg gaagtgtttt 28800 aatgcataaa atatcatcga caaatcatga aggttgcccc agcacctggg aatatagctg 28860 ggataagcca ttatgttttg gagtcaactc catgggtgga tatttaagct tctgaagatc 28920 ttcccctata tacaactctg cgagtaaatt catgaatgaa gcccatgtgt gacaagtggc 28980 tctccattat agctcactta caaatttagt agccaactga ttcaatgaaa ggaaaaagtc 29040 ctgcgggctt tttcaatacc cctgaacccc cctgttccca tttctgttga atcagaaatc 29100 actttaccta tctttgttgc attagcagaa acccagtcta aggtgacttc ctataactgt 29160 aaactttaca gatgttccct caagctggag gagaaggggt tgacaaaaca gagtgttttg 29220 tggctcctta aaagtcagcc tgcctttgaa gctttgaggc aaggtcctaa gcctgcagga 29280 aaatcagcct caggtcaaga gtttataaga gctcagttgc atggaatcag tactgcatga 29340 ggggaggagc ctgcagagtt ctcagggtct cagcaatagc tttttgaaaa acatctctgt 29400 gctggccagg cgcggtggct cacgcctgta atctcagcac tttgggaggc cgaggcgggc 29460 ggatcacgag gtcaagagat caagaccatc ttggctaaca ctgtgaaaca ctgtctctac 29520 taaaaataca aaaaaaaaat aaaaattagc caggcgtggt ggtgggcacc tgtagtccta 29580 gctactcggg aggctgaggc aggagaatgg catgaacccg ggaggcggag cttgcagtga 29640 gccgagatcg caccactgca ctccagcctc ggcgacagag ccaagactct gtcttaaaaa 29700 aaaaaacaaa gaaaaaaaaa gaaaaacatc tctgattcca gtaattaaaa attctatttc 29760 attccacgaa tatttatcag tgccacatgt gacactatgc agcccagcag ggatatagat 29820 aagcgtgagg aagacacagt tgctaacatt taaggacaga taaactaagg caggggttgg 29880 cacactggta cccacagtcc aaacctagct tgccaccagt ttttgttaac aaaattttat 29940 tgacacacag ccatgctcat tcatttatgt attgtctgtg gctgctttca caatacaaca 30000 gccaagtcga gtagttgtgt cagataccat acagcctgga aatactatct catcctcgat 30060 aaagtaactt tgccccaacc tctgctctag gggcaggatg ggcaagtgtc ccaatggcaa 30120 tatcaccaat agggccagaa gtgacaagca cagagcagac gttcgcaggg ctgtggagcg 30180 gggagggaga agccttcata tctttaaagg aaaccaggaa aacttcatgg accaaggctt 30240 caaagtgggc ctcaaaagat gggtaaaatt tctgcagata attgtttaga gattgggttt 30300 caggaagaga atatggcaag gacacgtggt cactgtataa gggaggcaat ctaagatgtg 30360 tcctagaaac tggagaatgg gctacaaaga aagcagcact aaggaacaat gctgcagagg 30420 gaaactgttg cagaatattg agggtgtcag tgagtttgta tgtaactgca agcagagagt 30480 cactggaggt tgggtagtaa caaaatagga ggtggctcag acatttccac atgcaaatag 30540 attcagtagt tcctttttag ttcaaatgaa ccttctattg cccttattag tgattctata 30600 aagtaaaatc tacacagtgc agagggtggc cttagaggct aacgagcctg gtttcctgcc 30660 tcagctgccc tcactgagtg taggggtgct ttctttaatc tctggaaacc tccactgtct 30720 catctgtaaa atggagataa tactaacacc ttgatgtgat gtcatgaaga gaaattaaga 30780 gaggcagtgt aagtaaagtc cccacataga gcctgggaca tacaagccac ttcataagtg 30840 tcaattctta ttgaaccttt ttattaagaa actaacaata ttctatcatt ctggacctac 30900 aaaagggcaa tttcatgtgg ctcaacttaa ggtttagggg aagcagtgag agaaatgaca 30960 acttgatgct tgtccattgt gacatgacag acctcttgac aagctaagac tcccattgtg 31020 atgagcctct cacacctggc cattccaatg gaacagacag ggtaaggacc aatctggact 31080 gtgttatctt ttccaggtgc aagtatgtgc tatgggtaag tgccagtttg gagaactccc 31140 ttagccacag gaaatgaaaa ttcatgtgat tgtttgaagg attcagcttc tctttgctgc 31200 taatccttgg gttttgtgca cctagaatgt ggtctcctgc aggccctgaa agccttgaat 31260 tcctggcatc tttgctgtga aggtctccct ggctgctgct ggaggaaggg gctggaagga 31320 gtgagtgtgt gcacaggttc agagttcagt cttcagacaa aaggagtgag ataaattgaa 31380 gacaagctgc cgatggtagt gcatggaact gctcaatgac cagcttcctt agcgaaaaca 31440 ttagcaacac attcaggcaa agggatgcga gaagttaagt actttgcaga aatatttgac 31500 aggccctgca aacactgagc aagaaaccat aggttctccc caattgcagg gatgcaagta 31560 acgtgaacac tttcctttcg gtcatcttcc ttggtggtca ggcatcatct ggatcacttt 31620 catctggcat cgggttataa ctacctgacc ctctcagact ggggtgaatg tatcatcttt 31680 ccaaggtgtt tgccgttccc aacaaagaga ggaagccagt tcgctattgg cctgttagct 31740 ttacaaacgg atggtagagc ttatgcttac caaggaagag tgaaagggga ttatcgacca 31800 cttgttgaca gggaaaatag tttaatcaaa ctgtaactca gctactcatg gccactgaga 31860 aatctgagaa agcctctgtc ataataacac acataataat cctagtatta gaaagccctg 31920 cgctctggct aagactctac tatacttttc agtaacttat ttccccagaa tctccatagg 31980 gatgcaattc cttcacccct gctttaagtt acttctctct cctcgcctca gtgatgtcat 32040 catatacacc tgtggacaaa agccgtgaca gggaaggaga tgccatttac gtccctggtg 32100 attctatagg aaactaaggg acctccttat cacccttcta tgaactatgc ccctgtcagc 32160 tttaaaaatt tgttgttgtt attccaattt tttttttttt gagatggagt ttcactcttg 32220 ttgcccaggc tggagtgcaa tggcacaatc tcggctcacc acaacctctg cctcccaggt 32280 tcaaaccatt ctcctgcctc agcctcctga gtagctggaa ttacaggcat gcgccaccac 32340 gctcggctaa ttttgtattt ttagtggaaa cgggttttct ccatgttgtt caggctggtc 32400 ttgaactcct gacctcaggt gatccgcccg cctcaggctc ccaaagtgct gggattacag 32460 gcatgagcca gcacacctga cctgttattc caatttaaca gttctttctt cccatacctg 32520 taaatgtgtg tgactgtgtg tgtgtgcatg tacactcaca cacacacaaa tacacaagtt 32580 caagtgaaat ctaaatgctt ggtaaaacag tccatgtgca ctaatttgca agagttgttg 32640 tgagggtaga gcttttgaat aaacataggt tgtcaaagga aaaactccct ctgtgtaagc 32700 cacaggacaa aggttttgaa acacctttgt tatctaaagc tggaaagaaa tgtcttgcct 32760 taaaagaatt tgcacattcg tacctctttc cacaaatacg tgaaaggacg tgcttttgaa 32820 gataagaaaa gtttaaattc tacaaaaaaa aaaaatctga tttgggcaga actcattgct 32880 cccttttctc tgtttctacc ttgttcttct ctgggtggat catttactac ttatactgtc 32940 agtgttggtg ttgcttgttt acttagatcc ctgaagtcga gttgcacaac tccagggggc 33000 attcagataa aatatcatgt gaatgatgcc ctggagtttt gcaggtagct ttgtcctgaa 33060 gacagggaca aaaatgtttc atctctttac ctcccagtgc ctggtggaca tgccttcgga 33120 ccaagtagtt gcacaattca ttgttgctta gcaaatgaac aaatatgttc acctcactaa 33180 atagctgaca tgaaaacatt ttaaaaatag tatcaagata tttaaacagt cgattttatg 33240 aatttaaaag acacctagag atactacaat tccgtagttt tttagattaa aaaacaaaga 33300 cccaaagtct atatttttta aaggaaggcc cagatggttt tgtgaaggtc atgtgagtat 33360 ttagggacaa aactagagct gaaactcaat tctcttggcc ccaggtgatc ttctcactcc 33420 accagactta ctcagttcac atcacagtca caattcagat tagagctatg aacaattcta 33480 tccatttgca caattctaac tggtgtttct aacttcatta aaagactctg aattattttt 33540 cttatatacc tctaatcaag atcatttggt attatcctgc atatgttcaa atgttaccta 33600 tctacagata tttgaactta ggtggggaat atctccacaa agtccattaa gtaagttcag 33660 ttttagtgaa aactgagatg gtgcagcttg agagattaag tgtagaattt ccaatgtaat 33720 gctttgaatg tgtaccttaa atctgtatca ctggcttatt ctgggaattg aagtcttatt 33780 tcatttctca gagaatgatg gttctgctac cagtaatctt taagggttag atcattcggg 33840 ttttttgttt gtttgagact aaataaatga agaaaacaca tgttagatac aaaacactag 33900 aaatatatta attttcactg gagcgacacc aaaggccatc gacattaaaa atgaactcct 33960 aagttctttg caattcccca ggtatagatt taatatacaa cacatgcatc tcttgaaact 34020 ctttctttgc tagtaagaat tattctcctg aaatacccac ctgtcaaaaa gaaaggtaac 34080 attattgatt tttagaattc ttatttctgt cgtgtcagta agcaataccg gaaagaaaat 34140 caaacactca ggagaattgg catgatggtg aaggttgagc ttacaagtac agtggactca 34200 agtatccatg atccagcgca ctgagcaata aatccaaatg agcagtgacc acaggaaaac 34260 aatatgcagg gaggcctcgc tgggaaaagc taaactttta tatatgggaa tagtctatgg 34320 aggattacag gggatgtttt cttgggggta taaggtctgg agtgtgcagt actgggtgaa 34380 gcccttatct aacaggcaac agaaaggtct tcccaggtta ggcacacgtg actctacctc 34440 caacacagaa tttttttttt taagaaagca acagaaattt gcaaatgata gtctggtctt 34500 ttgtcctctc aattttaaag caaataacca gtattgtgtt atctaccttt tcatggatgc 34560 atccagtgtg cctagaaggg ccagacttta ttctgtattt gcaacaaaag tagacccagc 34620 aactgatggg aagatatctg attgggaagc agaagcagct ggtattttaa atcaggatga 34680 aagctaagat tttaggactc acttttgata ggaagaaagg atatatcaat tttcctttta 34740 atgagtggga tttttgaggt actttgtggg gcctctggtt caagactgtg gccagtgtgg 34800 tgttgtagga ggggcactga tggagagcta tcccggtgca ttatttctga gccacctctg 34860 tgcactttac ttcctcattt gtaacatggg actaatgtgc cctgctgagt cctcagagtt 34920 gctgcaacaa tcaaatgagc tattaaggga taagctcttt tccagctacc tatgagaatg 34980 ggtatgatat ggtcccagaa tttgctctct agagccacag aaatctctta acccctacaa 35040 gaactcttta aagttgttat ccccattata tagatgagga aactgagact tagacaaaaa 35100 gttgtccaag atcacataat attaaggaac agagctggga tgaatatttg agtctaattc 35160 caaaacattc tggaataact caatatgtgg ttttccattt ctcccaaaaa caggtacctg 35220 cttttttcag tggcttgtgt tccagctgac agctccaggg cctgtttgaa taattcgaag 35280 acaatcctta gttaggaaag caagctttaa ttatcactgg ggaacagaag gccgcatctt 35340 cgaggaattt ggcagacctc agcagggggc aaccacaggc ctttggcaaa agatcacttt 35400 tcaacaacat tgtcaattcc agtgaccccc gaccttccac ctgcaggtcc ctgaacagct 35460 gctgttctgt gggaagcagt ggcagtctgt cttcctttaa aaggcacatg cacactctgt 35520 ccctgctgcc tgctgagatc ccacctggga cctcatcccc agagctgggg gtcatctccc 35580 atatcaagaa attaagaaaa ataagggggg gcaggaaagg acagctttga caacagtccc 35640 tgaactttcc cttttaatat aagccagatt taacgtatgt cattctgtaa atccgggagt 35700 ccaatttgag gcttgtaatt tgctgcaagc ttccctgttc ctccaagtgg ggtggagcta 35760 tgccaagcac atcaaggtaa ctggtggaag atataatttc cccactgtga gcctgcattt 35820 cagttcccta ttgtaatttt tatttgtgtt gaggttttgt ggttttaaaa aagtcaacca 35880 gatttatttt taaattaacc cagcccaaca tcaaaggcaa taagtagagg atgtttaggt 35940 attataaaga aaccctgtgt aatctgttat agctgtattc tttctcaggg catgtaatgg 36000 taaatggtta ggggcctttc acaaccaact ttctatattt ctctgacctc ggactaccct 36060 catgggcaaa aaaccctttt tgaggggatt tagtagcccc tctctcctcc tcctcaacct 36120 cttaatctaa tcctgtttgt aacgcaacat gctgcatgaa gaatacgaaa catgggctta 36180 agtccctccc cacttccctt atgactgtgg tttactttta gatatgaaga actctttcag 36240 gccaaaaaaa aaaaaagggg gggaccattt ggttaacgaa ccattttctt tggtaggcag 36300 gagaaagttt atattgaaag tttatcttaa ggatgacagg tcatacctga agggtttgtt 36360 ttggaatact gtggattttt tctaacccaa ataattacaa gagagttcct tgtttattgg 36420 ctcatggagg aaattcaagc gcctctgttc taggcatttt aagtgctctg tatatatggt 36480 ggttgttcct caaaacagct ttgggtttgt tttttgtttt ttgttgttag tggtggtttt 36540 ttgagataga gtctcgctct gttacccagg ctggagtgca gtggcgcaat ctcggctcac 36600 tgcaccctcc acctctctgg ttcaaaagat tctcctgcct cagcctcctg agtagctggg 36660 attacaggcg cccgccacca tgcccagcta atttttgtat ttttagtaga gacggggttt 36720 ccccatgttg gccaggctgg tctcgaactc ctgacctcag gtgattcacc cgcctgggcc 36780 ttccaaaatg ctgggattac aggcgtgagc caccatggct ggcccaaaac agctttttaa 36840 gaaaagtgct attaacccca tttacagatg agcacatttg agccacaccc tcttttccac 36900 actctaaatc ttgtctcttt ctttaaacat gagttactta tacttctgag cataactgag 36960 gcacttttag agacagtgtc ttctaagctc aatgtgatat tatttgtgct gctgtgctgc 37020 tactgggtaa ccagcaccca tcctggtcac cagggtaact ttgtcaacca agaaggccaa 37080 ggatcccaaa ccagcatttt ctactatcaa aagagaggtt ctgcaaatcc actggcagga 37140 gagaaaatat aatagcaggt ggcatttata tgacccagtg tgcatggcag tgtcccaggg 37200 tatcaccgtg aatctcagaa actccaggct ttccccatgg gaaatccaca ccaccacaga 37260 tccagtggag gactcggtca agactcctga aatcaaagaa ctcacagtga ctgattcttt 37320 ccctagtttt ataatataaa taatggcatg gggtcacatt cagccgtcat tatccacatc 37380 atttcactga tgggatcctc ctcagacaga gattgggaat cagatttctc ggtcacataa 37440 actgttgctc attctgtgag gctgcctatt tgtaaagttg tggttcttat taaaagcaat 37500 ctcagacgta gcaagcaccc ctcacttccc ttctcattca ttttgttaaa gcaaatgggc 37560 tttggaattc aggcctgttt ctaccactta ctaatgttgt taatttggag gagttcctca 37620 actttgccaa ggcttgattt tctctgctgt aaagagggaa taataaacct attttacaga 37680 gcagctgaga caattaggtg agttaatgta tataaaatgg tttgcataat acccaacaca 37740 tattaaactc tcactcggtt tttaatatta acctctatgt gcttaataac attgaagaag 37800 aagattcaag tagattatag tctgttaaag agttcaaata taaataaata attctcaggg 37860 tgagaattgc catagcatag atatgttacg tacccatggc agagcgtgag gtggcagcat 37920 ctaatggttg agggagttgg gtgagacatc aagagaaggt gacatatttt tttgagtacc 37980 cagtggaagg catgggatac catgtggatc tctgcagtag attaaatata gacttgaact 38040 aacctatcct ggaacaatag gacaatatcc ttgtggctta cagtaattat tccctgcacc 38100 tatattgatt tgtttattaa acgaatagct ttattggtaa acatgtatat tgcggaagta 38160 gacttggtta tcattcccac aagtccagtt aaagtaatgg catctatata aaaaactcat 38220 aaaaactaga tatgtaagta atcaataaaa tactcttctc aagtattcag gagaaaaaat 38280 gtgttgaaat gatgattcat cattccacat aacgtatttg tgactacatt taatagcctc 38340 attagcaata aaatttttat gagttaacat catatgagaa tattcccttg taccttaccg 38400 agactttatc tgtagatttg taacataacc ataatcatct tggtatgttt cttacacatt 38460 ttattcagtg aacccaaatg aacttctaat tacatgttca gctgccagtc atggttttat 38520 atgtttgaat atatatacct tcagaggata tttgctcttt ggggtggtga agacttcatc 38580 ttcttataaa tgcaaacaga agatagttgg aagaggaaaa tgttttagca gtgtctcaat 38640 tatctctcct taatgattat ttcacaacct cgagatattt tcctaaaaga ctaagtaaga 38700 aatatatagt aagattcctt tctggatatt ttcagaactc ctagttataa ctatgcacca 38760 aatatggata aacactggat tatgcagtac acaggaccaa tgctgcccat ccacatggaa 38820 tttacaaaca ttctacagcg caaaaggctc cagactttga tgtcagtgga tgattctgtg 38880 gagagggtaa gcacatgaac ctacctcagt gatagttttt ggcccagctt cctttgtgta 38940 gacttattct tgccaatcct gtttggtttt ttccccttca ttttccagca tcattttgag 39000 agagaaagaa agagagagag tatgtgttta gtggcttaat catccctccc ttatcttgtc 39060 ctcattccat ctacctctcc agggttggtt tcttatggag ccagtaaaaa agaggagaga 39120 aaaatcaaat cagcgtagat caggggccac atcctcaaag gcaataaaga attgatggag 39180 cttgtgctga acttgaactt taagttaagg gccccatcta aaggaacagc aattactcag 39240 ctccagctaa attttgccat gtagaaatgt ggatcaagta tcagcaggtc ttctgacttt 39300 tttaaagaag ccagaaacac aaaaaatttt atttgaaatt ttctgaactt tgaaacatag 39360 tataagccaa acaagacgtg cctcaggctg gatttaactg gatcaggctc agaagcagcc 39420 tgtttttaac ccttggtaat tagatatgtg atgataattt taacaatgga ttttcaaagt 39480 acaacctata aagtttgatg gtagaggttg ttgtgcgggg tgtttttgtt tttgttctac 39540 caaacaagca aacaaaaagc ctaaaagtag aatgtgctag attccaaaaa gttacatttc 39600 acctttacca ttggaccttt ccctcccaga ctgtaagcaa atagaaaatg tggataatgt 39660 tattaaagca actcttgcct tttaaaaata acaggaaaaa gatttggggg caatcgtggc 39720 aacactattg agcatcatct tataccagca caattgattc tgacttgttc ctttgctgta 39780 tttcagctgt ataacatgct cgtggagacg ggggagctgg agaatactta catcatttac 39840 accgccgacc atggttacca tattgggcag tttggactgg tcaaggggaa atccatgcca 39900 tatgactttg atattcgtgt gccttttttt attcgtggtc caagtgtaga accaggatca 39960 atgtacgtat ttctctgttt gcaacattca actgtcgtac ctcaagtgtg tctaagataa 40020 ttcaattacc agtctcagta tctggtttcc tttcatccaa aacaaaaaag gatgtgtgta 40080 ggctggttaa tttcgaagat gaaaaccttt tcctccctgc cacatcttaa attagctcaa 40140 gtatactact taaagagaaa ggaaaaataa gtgtatcaat gactaattct ctcaaattga 40200 ctggaatcta tgtctttttg gtctgtgtgc acagacagga tgtgatcttc tgggatatca 40260 cccttctttg aatcagagat acgctgtcat ttaaaaaaaa aacctgacac catcctttta 40320 gtgtttaact tttaaaaatt attccgaaag aaatgttttt aaaagataaa ttttgaaaag 40380 ctggcttttc ttttaaagga aaaagagcta aaggactagg ctgctatttc tgtcactgta 40440 ggcaggtcac tgcttctctt tgcatctcta ttttcccatc atgaaatggc cttgcctatt 40500 ttcccatcat aaaatggcct tgtcaatcat ctcaggatgt tttgaataaa atgggattgc 40560 atccatgaaa gaattatgga aagactaaaa gaaaaagtgg aagtagaatc cagaagctgg 40620 aatggccctt gaaaagcatc tagcctggac ccctgatgta acagtgaagc taggcagaca 40680 gcgtcagtgc ccctctcaga tccctgattc tggaagtggt ggcagtgggg cccagagccc 40740 agatgttctg aatctccccc gagtaccaca tgtgctgctg catcatacta catcctggag 40800 agagggcaat tgaagtaaga aaggtttctt tgaagaatgt tattgtgctt cctaagatta 40860 ttagaaacac cctgagaatt gtcagatcta tatctagaag gctttatgtt ttaaggacta 40920 gatagcatag attgaattca actgtaaaaa ctgtacatcc ttttttaaaa tattgatttt 40980 aaagctcttg ttcaacggaa aagttatgga ctattgggtt ttaagcaaaa gtatgcttgc 41040 tgcaaaaacc atgtatcagg ctgcatcttg cctggtgatg tggtcagaat acaggggtgc 41100 aggcatctct ccagcctgac cctggcaaga gtcagttaat cttgctcagt gccattgctg 41160 tgatcacaca cccacccttg ccacacaact acccatgcct aggagaccag atgagagggt 41220 gagaagagtt gaaggccaat gagtcactgc tgtagaaaaa gcagccctaa gtgccacctt 41280 cccctggcat tggatctcag ccatcaccgt gtgccccttt acagagtccc acagatcgtt 41340 ctcaacattg acttggcccc cacgatcctg gatattgctg ggctcgacac acctcctgat 41400 gtggacggca agtctgtcct caaacttctg gacccagaaa agccaggtaa caggtgtgtc 41460 attgttcctc ctctcagcca gccccaaata cactgagctc cagctggtgc ccagagccag 41520 ccagcagctg aagacatgga ggcagaatat gccttgccca caaggatcac cccaagctga 41580 gcatttctca gctgcttgtg aatagcatat tgatggagat gcactcatgg tctgtgggaa 41640 gtgagaggtg tttctttaaa taagctgtta gcacagatcc atttggaaaa acgtccagat 41700 gccaaaagta aatattatca ttttgctttc aggtttcgaa caaacaagaa ggccaaaatt 41760 tggcgtgata cattcctagt ggaaagaggg taattattgg ttcctggggt gcttctggga 41820 accagtccta gtgggcagct ttccctgctg agtatttttt ttctccttat ttttgtttac 41880 taagcatgca gatttcgtaa acctagtcac aagattgaat ggtttgctgc ttattctgta 41940 gtggtcaata gagtaataat tgctggatca gaattgtaaa gaataaccct caagttggtt 42000 aattggtaca aaaacacagt tagatagaag ttatagaatt tgatagtata gttgggacat 42060 tatcgttaac aataatttat gtatatctta aaatagctag aagtgaagaa ttgcaaagtt 42120 cccaacacaa ggaaaagata aatgagatga tgaatatccc aattatcttg atttgatcat 42180 tacacattgt agactggtat ccatatatca cacgtacccc caaaatatgt ataattgtga 42240 tatatcaatt tttaaaatac caaaaaagca agagaatgac gactccacat cccccaaaaa 42300 gaataaattc tcataagctt ggaccaaagc ctttatcatg ggtgtagatt tactgttgca 42360 tttctcagtg ctggtttcta atcagaccag tggattgagt ttctctacca tcctccccac 42420 gttcttctct aagctgcctc caagcctcac ccggcaccct tcttcctact tcctacttct 42480 tttccttgtg tgcctttcct agttttaaat agataaatgt atgccattgt aattatttcc 42540 attgtcactt ctgggtttcc ccttttggtt c 42571 4 360 PRT Human 4 Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu Gly Thr Glu 1 5 10 15 Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25 30 Arg Ile Gln Gln Glu Arg Lys Asn Ile Arg Pro Asn Ile Ile Leu Val 35 40 45 Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser Leu Gln Val Met Asn 50 55 60 Lys Thr Arg Lys Ile Met Glu His Gly Gly Ala Thr Phe Ile Asn Ala 65 70 75 80 Phe Val Thr Thr Pro Met Cys Cys Pro Ser Arg Ser Ser Met Leu Thr 85 90 95 Gly Lys Tyr Val His Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys 100 105 110 Ser Ser Pro Ser Trp Gln Ala Met His Glu Pro Arg Thr Phe Ala Val 115 120 125 Tyr Leu Asn Asn Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140 Asn Glu Tyr Asn Gly Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu 145 150 155 160 Gly Leu Ile Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170 175 Gly Ile Lys Glu Lys His Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180 185 190 Asp Leu Ile Thr Asn Glu Ser Ile Asn Tyr Phe Lys Met Ser Lys Arg 195 200 205 Met Tyr Pro His Arg Pro Val Met Met Val Ile Ser His Ala Ala Pro 210 215 220 His Gly Pro Glu Asp Ser Ala Pro Gln Phe Ser Lys Leu Tyr Pro Asn 225 230 235 240 Ala Ser Gln His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Met Asp 245 250 255 Lys His Trp Ile Met Gln Tyr Thr Gly Pro Met Leu Pro Ile His Met 260 265 270 Glu Phe Thr Asn Ile Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser 275 280 285 Val Asp Asp Ser Val Glu Arg Leu Tyr Asn Met Leu Val Glu Thr Gly 290 295 300 Glu Leu Glu Asn Thr Tyr Ile Ile Tyr Thr Ala Asp His Gly Tyr His 305 310 315 320 Ile Gly Gln Phe Gly Leu Val Lys Gly Lys Ser Met Pro Tyr Asp Phe 325 330 335 Asp Ile Arg Val Pro Phe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly 340 345 350 Ser Ile Val Pro Gln Ile Val Leu 355 360 5 307 PRT Human 5 Asp Val Glu Leu Gly Ser Leu Gln Val Met Asn Lys Thr Arg Lys Ile 1 5 10 15 Met Glu His Gly Gly Ala Thr Phe Ile Asn Ala Phe Val Thr Thr Pro 20 25 30 Met Cys Cys Pro Ser Arg Ser Ser Met Leu Thr Gly Lys Tyr Val His 35 40 45 Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys Ser Ser Pro Ser Trp 50 55 60 Gln Ala Met His Glu Pro Arg Thr Phe Ala Val Tyr Leu Asn Asn Thr 65 70 75 80 Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu Asn Glu Tyr Asn Gly 85 90 95 Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu Gly Leu Ile Lys Asn 100 105 110 Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn Gly Ile Lys Glu Lys 115 120 125 His Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr Asp Leu Ile Thr Asn 130 135 140 Glu Ser Ile Asn Tyr Phe Lys Met Ser Lys Arg Met Tyr Pro His Arg 145 150 155 160 Pro Val Met Met Val Ile Ser His Ala Ala Pro His Gly Pro Glu Asp 165 170 175 Ser Ala Pro Gln Phe Ser Lys Leu Tyr Pro Asn Ala Ser Gln His Ile 180 185 190 Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Met Asp Lys His Trp Ile Met 195 200 205 Gln Tyr Thr Gly Pro Met Leu Pro Ile His Met Glu Phe Thr Asn Ile 210 215 220 Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser Val Asp Asp Ser Val 225 230 235 240 Glu Arg Leu Tyr Asn Met Leu Val Glu Thr Gly Glu Leu Glu Asn Thr 245 250 255 Tyr Ile Ile Tyr Thr Ala Asp His Gly Tyr His Ile Gly Gln Phe Gly 260 265 270 Leu Val Lys Gly Lys Ser Met Pro Tyr Asp Phe Asp Ile Arg Val Pro 275 280 285 Phe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly Ser Ile Val Pro Gln 290 295 300 Ile Val Leu 305 6 309 PRT Drosophila melanogaster 6 Arg Pro Asn Ile Ile Leu Ile Leu Thr Asp Asp Gln Asp Val Glu Leu 1 5 10 15 Gly Ser Leu Asn Phe Met Pro Arg Thr Leu Arg Leu Leu Arg Asp Gly 20 25 30 Gly Ala Glu Phe Arg His Ala Tyr Thr Thr Thr Pro Met Cys Cys Pro 35 40 45 Ala Arg Ser Ser Leu Leu Thr Gly Met Tyr Val His Asn His Met Val 50 55 60 Phe Thr Asn Asn Asp Asn Cys Ser Ser Pro Gln Trp Gln Ala Thr His 65 70 75 80 Glu Thr Arg Ser Tyr Ala Thr Tyr Leu Ser Asn Ala Gly Tyr Arg Thr 85 90 95 Gly Tyr Phe Gly Lys Tyr Leu Asn Lys Tyr Asn Gly Ser Tyr Ile Pro 100 105 110 Pro Gly Trp Arg Glu Trp Gly Gly Leu Ile Met Asn Ser Lys Tyr Tyr 115 120 125 Asn Tyr Ser Ile Asn Leu Asn Gly Gln Lys Ile Lys His Gly Phe Asp 130 135 140 Tyr Ala Lys Asp Tyr Tyr Pro Asp Leu Ile Ala Asn Asp Ser Ile Ala 145 150 155 160 Phe Leu Arg Ser Ser Lys Gln Gln Asn Gln Arg Lys Pro Val Leu Leu 165 170 175 Thr Met Ser Phe Pro Ala Pro His Gly Pro Glu Asp Ser Ala Pro Gln 180 185 190 Tyr Ser His Leu Phe Phe Asn Val Thr Thr His His Thr Pro Ser Tyr 195 200 205 Asp His Ala Pro Asn Pro Asp Lys Gln Trp Ile Leu Arg Val Thr Glu 210 215 220 Pro Met Gln Pro Val His Lys Arg Phe Thr Asn Leu Leu Met Thr Lys 225 230 235 240 Arg Leu Gln Thr Leu Gln Ser Val Asp Val Ala Val Glu Arg Val Tyr 245 250 255 Asn Glu Leu Lys Glu Leu Gly Glu Leu Asp Asn Thr Tyr Ile Val Tyr 260 265 270 Thr Ser Asp His Gly Tyr His Leu Gly Gln Phe Gly Leu Ile Lys Gly 275 280 285 Lys Ser Phe Pro Phe Glu Phe Asp Val Arg Val Pro Phe Leu Ile Arg 290 295 300 Gly Pro Gly Ile Gln 305 

That which is claimed is:
 1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a protein comprising the amino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consisting of SEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and (d) a nucleotide sequence that is completely complementary to a nucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising a nucleic acid molecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4. A process for producing a polypeptide comprising culturing the host cell of claim 3 under conditions sufficient for the production of said polypeptide, and recovering said polypeptide from the host cell culture.
 5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.
 8. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the protein of SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence. 